PROJECT SUMMARY Traumatic brain injury (TBI) is a very common problem with over 2 million new cases requiring medical attention annually in the US. A neuropathology that is widely shared among various types of TBI and across spectrum of severity is diffuse or traumatic axonal injury (TAI), a form of ultra-rapid deformation and disruption of axons caused by rotational acceleration and shearing of brain tissue. In this project we use a visual model of TAI and explore the hypothesis that the DLK-JNK signaling cascade, known to be activated in simple axotomy lesions like nerve crush, triggers downstream degenerative effects of TAI in neurons projecting in the optic nerve, i.e. retinal ganglion cells (RGCs). We ask three questions, in logical order: is DLK-JNK pathway activated in visual TAI? If so, does DLK-JNK activation trigger retrograde degenerative effects in RGCs as in simple axotomy models? And does blocking of the DLK-JNK pathway prevent or treat RGC degeneration and related system impairments such as disconnection with CNS targets and loss of vision? To inflict TBI, we use the impact acceleration model of diffuse TAI with which we have consistently produced primary optic nerve injury in our laboratories. Our experimental subjects are conditional knockout mice for key members of the dual leucine zipper kinase (DLK-JNK) cascade. We also employ CRISPR strategies to disrupt the initiating kinases of the DLK-JNK cascade, DLK and LZK, and also treat wild-type injured mice with the DLK inhibitor sunitinib. Our experimental readouts include kinase activation, cell death, axonal and terminal degeneration using conventional and high-resolution (CLARITY) neuroanatomical methods, and behavioral measures of visual acuity. Besides exploring the role of key members of the DLK-JNK signaling pathway in perikaryal and axonal degeneration in visual TAI, the proposed experiments provide proof of concept for the protective or therapeutic effect of DLK/LZK blockade in TAI. The project leverages the complementary strengths of our laboratories and the design and experimental methods are supported by extensive preliminary studies. In Specific Aim 1 that serves as the foundation of the proposal, we will determine whether TAI in the visual system is associated with activation of the DLK-JNK signaling cascade in RGCs and we will identify key signaling molecules. In Specific Aim 2, we will explore whether blocking JNK and DLK/LZK signaling with knockout/genome editing strategies or pharmacological inhibition prevents or aborts RGC perikaryal and axonal degeneration in visual TAI. In Specific Aim 3, we will determine whether blocking DLK/LZK activation with knockout/genome editing strategies or pharmacological approaches prevents or aborts neural system impairments (visual disconnection and dysfunction). Taken together, these experiments examine the role of the DLK-JNK pathway in neuronal degeneration, disconnection, and dysfunction following TAI in a model CNS system and explore novel protective and therapeutic strategies for diffuse TBI.